Myeloid Leukemia

78 Branford and Hughes
3.3.1. PCR
1. Using the Primer Express software, primers were designed to amplify a region
flanking the primers that are used in the quantitative assay. These primers produce
a PCR fragment that is larger than that used for the quantitative assays and
includes the same sequence.
Primers for the BCR standard:
Forward primer: 5� tca cca aga gag aga ggt cca a
Reverse primer: 5� gtc tga aag agc gat gcc ct
Primers for the b2a2 standard:
Forward primer: 5� aga agc ttc tcc ctg aca tc
Reverse primer: 5� aga tgc tac tgg ccg ctg aa
2. Extract RNA from the b2a2 expressing cell line and prepare cDNA using the
procedures outlined under Subheadings 3.1. and 3.2.
3. Add 2.5 µL of cDNA to a 0.2-mL tube containing 2.5 µL of 10X PCR buffer, 5 µL of
25 mM MgCl 2, 0.5 µL of 10 mM dNTP mix, 0.1 µL of 50 µM forward and reverse
primers, and 0.5 µL of 5 U/µL of AmpliTaq gold. Add sterile water to 25 µL.
4. Transfer the reactions to the thermal cycler and amplify with the following conditions:
a. 1 cycle at 95°C for 12 min.
b. 30 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s.
c. 1 cycle at 72°C for 7 min.
5. Assess the size of each amplification product by electrophoresis of 5 µL mixed
with 1 µL gel loading buffer on a 2.0% agarose gel using the Puc19 size marker.
The b2a2 fragment is 127 bp and the BCR fragment is 293 bp.
3.3.2. Cloning
1. Purify the remaining PCR product using the UltraClean PCR Clean-up DNA
purification kit according to the manufacturer’s instructions.
2. 100 ng of the purified PCR product is cloned using the pGEM-T Easy Vector
System II kit according to the manufacturer’s instructions.
3. Prepare the plasmid DNA using the Qiagen Plasmid midi kit according to the
manufacturer’s instructions.
4. The presence of the cloned insert is determined by PCR amplification as under
Subheading 3.3.1., followed by sequencing to confirm the correct sequence.
5. Linearize the plasmid by digestion with Sca1 restriction enzyme.
6. Determine the concentration of the plasmid DNA by assessing absorbance at 260 nm
and 280 nm. The copy number of the plasmid is calculated from the concentration
and size of the plasmid (vector size + insert size [bp]), and using Avogadro’s
number of 6.02336 × 10 23 molecules in 1 mole of template (6.02336 × 10 14 molecules
in 1 nmole). Using the b2a2 plasmid as an example:
a. Calculate the molecular weight of the plasmid by multiplying the size by 660
(average molecular weight of double stranded DNA) = 3129 × 660 = 2,065,140.
b. Therefore, 1 mole of template = 2,065,140 g and 1 m mole of template =
2,065,140 ng.